Series of Skin-Whitening (Lightening) Compounds

ABSTRACT

The present invention is directed to inhibitors of tyrosinase, pharmaceutical compositions comprising such tyrosinase inhibitors, and methods of making and using the same. Specifically, included in the present invention are compositions of matter comprised of at least one 2,4-dihydroxybenzene analog, which inhibit the activity of tyrosinase and which inhibit the overproduction of melanin.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/082,422, filed Jul. 21, 2008, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to novel tyrosinase inhibitors,pharmaceutical, dermatologic and cosmetic compositions comprising suchinhibitors, and methods of making and using the same.

BACKGROUND OF THE INVENTION

The color of human skin is determined by the amount and the type ofmelanin produced by specialized cells, melanocytes, which are located inthe basal layers of skin. Melanin, one of the most widely distributednatural pigments, is a homogeneous polyphenol-like biopolymer with acomplex structure and color varying from brown to black (eumelanin) andred to yellow (pheomelanin) (Prota G. Med. Res. Rev. 1988, 8, 525-556).In human skin, melanin is believed to act as a protective agent againstultraviolet radiation. As such, people living close to equator havedarker skin than those living in areas away from the equator.

Overproduction of melanin can causes different types of abnormal skincolor, hair color, and other dermatological disorders, such as melasma,age spots and sites of actinic damage. Melanin is produced through aseries of oxidative reactions and polymerization steps starting with theamino acid tyrosine. Tyrosinase (EC1.14.18.1), a type III,copper-containing enzyme catalyzes two initial reactions in theproduction of melanin: 1) the ortho-hydroxylation of L-tyrosine bymonophenolase action, and 2) the oxidation of 3,4-dihydroxyphenylalanine(L-DOPA)→o-dopaquinone by diphenolase action. The later oxidation stepis much more rapid than the former, thus, the hydroxylation of tyrosineis considered to be the rate-determining step in melanin biosynthesis.Subsequent conversion of o-dopaquinone to melanin occurs through aseries of enzymatic and non-enzymatic polymerization reactions. Otherenzymes, such as dopachrome tautomerase (Tyrosinase Related Protein 2;TRP-2) and dihydroxyindole carboxylic acid (DICHA) oxidase (TyrosinaseRelated Protein 1; TRP-1) are also involved in the process of thebiosynthesis of melanin. Since tyrosinase plays the key role in theprocess of melanin production, inhibitors of this enzyme are often usedas skin-whitening agents (Mosher et al. In Dermatology in GeneralMedicine, 1983, 205-125, Fitzpatrick T. B., Eisen A. Z., Wolff K.,Freedberg I. M., Austern K. F. (eds), Mc-Graw-Hill, New York; Maeda K.,Fukuda M, In vitro effectiveness of several whitening cosmeticcomponents in human melanocytes. J. Soc. Cosmet, Chem. 1991, 42,361-368).

A number of naturally occurring, as well as, synthetic tyrosinaseinhibitors have been described in the literature. The majority ofcompounds comprise a phenol structure. These compounds act as metalchelating agents (Kojima et al. Biol. Pharma. Bull. 1995, 18, 1076-1078;Seo et al. J. Agric. Food Chem. 2003, 51, 2837-2853; Fu et al. J. Agric.Food Chem. 2003, 53, 7408-7414; Kim, Y.-J.; Uyama, H. Cell. Mol. LifeSci. 2005, 62, 1707-1723 and references cited therein). A pharmaceuticalproduct containing hydroquinone (2-4%) is moderately efficacious, buthydroquinone is considered to be cytotoxic to melanocytes andpotentially mutagenic to mammalian cells. Unfortunately, severalpurportedly active agents, e.g., arbutrin and kojic acid, among othershave not been demonstrated yet to be clinically efficacious whencritically analyzed in carefully controlled studies (Frenk, E. InMelasma: New Approaches to Treatment; Martin Dunitz: London, 1995, 9-15;Dooley, T. P. In: Drug Discovery Approaches for DevelopingCosmeceuticals: Advanced Skin Care and Cosmetic Products; Hori, W., Ed.;International business communications: Southborough, MA, 1997; Dooley,T. P. J. Dermatol. Treat. 1997, 7, 188-200).

Many tyrosinase inhibitors are resorcinol derivatives or polyphenolderivatives of flavonoids or of trans-stilbene, such as resveratrol orits derivatives. These types of compounds are known to form strongchelates with metal ions. (Seo et al. J. Agric. Food Chem. 2003, 51,2837-2853; Fu et al. J. Agric. Food Chem. 2003, 53, 7408-7414; Kim,Y.-J.; Uyama, H. Cell. Mol. Life Sci. 2005, 62, 1707-1723 and referencescited therein; Lerch, K. In: Metal ions in Biological Systems, pp.143-186; Sigel, H., Ed.; Marcel Dekker, N Y, 1981; Wilcox et al. In:Substrate analogue binding to the coupled binuclear copper active sitein tyrosinase, J. Am. Chem. Soc. 1985, 107, 4015-4027; Sanchez-Ferrer etal. Biochim. Biophys. Acta 1995, 1247, 1-11; Decker et al. Angew. Chem.Int. Ed. 2000, 39. 1591-1595; Decker et al. Angew. Chem. Int. Ed. 2006,45. 4546-4550; Briganti et al. Pigment Cell Res. 2003, 16, 101-110).Several compounds based on the resorcinol moiety have been used astyrosinase inhibitors, see: JP 2008-056651 A; JP 2000-095721 A; US2005/0267047A1; U.S. Pat. Nos. 5,339,785; 6,093,836; US 2008/0032938 A1;U.S. Pat. No. 7,282,592 B2; U.S. Pat. No. 7,339,076 B1; U.S. Pat. Nos.5,880,314; 6,852,310 B2; 6,077,503; US 2005/0271608 A1; U.S. Pat. No.5,523,421; US 2007/0098655 A1; US 2005/0267047 A1.

Although many compounds have been reported as potent tyrosinaseinhibitors, very few of them have shown skin-whitener properties.Additionally, most of these agents were found either to be toxic, orshown to have adverse side effects in humans. As such, the search fornew natural products or synthetic compounds having potent tyrosinaseinhibitory activity with low cytotoxicity continues.

SUMMARY OF THE INVENTION

The instant application provides a new series of novel depigmentingagents having excellent inhibitory activity against tyrosinase and verylow cytotoxicity. The compounds are stable and easily synthesized fromthe commonly available starting materials. More specifically, thepresent invention provides novel 2,4-dihydroxybenzene derivatives, whichare useful as tyrosinase inhibitors. The invention encompassespharmaceutically acceptable salts of these tyrosinase inhibitors. Alsoencompassed by the invention are pharmaceutical compositions comprisedof at least one tyrosinase inhibitor of the invention and at least onepharmaceutically acceptable carrier. The compositions of the presentinvention can be prepared in any suitable pharmaceutically acceptabledosage form.

The present invention also provides a method for inhibiting the enzymetyrosinase in a subject in need thereof, said method comprisingadministering an effective amount of a composition comprising at leastone 2,4-dihydroxybenzene tyrosinase inhibitor of the instant invention.

The present invention further provides a method for the prevention andtreatment of diseases and conditions related to the activity of theenzyme tyrosinase. The method of prevention and treatment according tothis invention comprises administering internally or topically to asubject in need thereof a therapeutically effective amount of at leastone novel 2,4-dihydroxybenzene tyrosinase inhibitor of the instantinvention.

The present invention also provides a method for inhibiting thesynthesis of melanin in a subject in need thereof. Such a methodcomprises administering an effective amount of a composition comprisingat least one 2,4-dihydroxybenzene compound of the instant invention.

The present invention further provides methods for the prevention andtreatment of diseases and conditions related to the overproduction oruneven distribution of melanin, said method comprising administeringinternally or topically to a subject in need thereof a therapeuticallyeffective amount of at least one at least one 2,4-dihydroxybenzenecompound of the instant invention. Diseases and conditions related tothe overproduction or uneven distribution of melanin include, but notlimited to hyper pigmentation spots caused by skin aging, melasma, liverdiseases, thermal burns and topical wounds, skin pigmentation due toinflammatory conditions caused by fungal, microbial and viralinfections, vitilago, carcinoma, melanoma, as well as other mammalianskin conditions.

The method can also be used for preventing and treating skin darkeningand damage resulting from exposure to the sun, ultraviolet (UV)radiation, chemicals, heat, wind and dry environments. Finally, themethod can be used for preventing and treating wrinkles, saggy skin,lines and dark circles around the eyes, soothing sensitive skin andpreventing and treating dermatitis and other allergy related conditionsof the skin.

In specific embodiments, the dose of the 2,4-dihydroxybenzene tyrosinaseinhibitor(s) of the instant invention administered to the subject inneed thereof is an efficacious, nontoxic quantity generally selectedfrom the range of 0.001% to 100% based on total weight of the finalformulation, and/or 0.01 mg to 200 mg per kilogram based on the bodyweight of the subject. Persons skilled in the art using routine clinicaltesting are able to determine optimum doses for the particular ailmentbeing treated. The compositions of this invention can be administered byany method known to one of ordinary skill in the art. The modes ofadministration include, but are not limited to, enteral (oral)administration, parenteral (intravenous, subcutaneous, andintramuscular) administration and topical application. The method oftreatment according to this invention comprises administering internallyor topically to a patient in need thereof a therapeutically effectiveamount of at least one 2,4-dihydroxybenzene of the instant invention. Ina preferred embodiment the composition is administered topically.

Included in the present invention are novel methods for the synthesis ofthe 2,4-dihydroxybenzene tyrosinase inhibitor(s) of the invention asdescribed in Examples 1-5. Thus, the present invention providescommercially viable options for the synthesis, and/or isolation,purification and formulation of the 2,4-dihydroxybenzenes of the instantinvention to yield a composition of matter having the desirablephysiological activity.

In yet another embodiment, the 2,4-dihydroxybenzenes can be used in thefood industry to inhibit the browning and color changes in fruits,vegetables, juices and other food products.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publiclyavailable publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed. Other objects,advantages, and novel features will be readily apparent to those skilledin the art from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates graphically a profile of the inhibition in theproduction of melanin (●) by 4-(pyridin-ylmethyl)benzene-1,3-diol (7) asdescribed in Example 7. The data is presented as melanin content (% ofuntreated control) vs. inhibitor concentration (μM). The IC₅₀ ofcompound 7 was determined to be 14 μM. FIG. 1 also illustratesgraphically cell viability (LD₅₀) (x) at varying concentrations ofcompound 7.

FIG. 2 illustrates graphically a profile of the inhibition in theproduction of melanin (●) by4-(2-(benzo[d][1,3]dioxol-5-ylethyl)benzene-1,3-diol (15) as describedin Example 7. The data is presented as melanin content (% of untreatedcontrol) vs. inhibitor concentration (μM). The IC₅₀ of compound 15 wasdetermined to be 3.3 μM. FIG. 2 also illustrates graphically cellviability (LD₅₀) (x) at varying concentrations of compound 15. The LD₅₀for compound 15 was determined to be 66 μM.

FIG. 3 illustrates graphically a profile of the inhibition in theproduction of melanin (●) by 4,4′-(ethane-1,2diyl)dibenzene-1,3-diol(16) as described in Example 7. The data is presented as melanin content(% of untreated control) vs. inhibitor concentration (μM). The IC₅₀ ofcompound 16 was determined to be 1.7 μM. FIG. 3 also illustratesgraphically cell viability (LD₅₀) (x) at varying concentrations ofcompound 16. The LD₅₀ for compound 16 was determined to be 260 μM.

FIG. 4 illustrates graphically a profile of the inhibition in theproduction of melanin (●) by 4-(2,4-dimethoxyphenethyl)benzene-1,3-diol(17) as described in Example 7. The data is presented as melanin content(% of untreated control) vs. inhibitor concentration (μM). The IC₅₀ ofcompound 17 was determined to be 11.9 μM. FIG. 4 also illustratesgraphically cell viability (LD₅₀) (x) at varying concentrations ofcompound 17. The LD₅₀ for compound 17 was determined to be 157.8 μM.

FIG. 5 illustrates graphically a profile of the inhibition in theproduction of melanin (●) by 4-(3,5-dimethoxyphenethyl)benzene-1,3-diol(18) as described in Example 7. The data is presented as melanin content(% of untreated control) vs. inhibitor concentration (μM). The IC₅₀ ofcompound 18 was determined to be 2.97 μM. FIG. 5 also illustratesgraphically cell viability (LD₅₀) (x) at varying concentrations ofcompound 18. The LD₅₀ for compound 18 was determined to be 222.2 μM.

FIG. 6 illustrates graphically a profile of the inhibition in theproduction of melanin (●) by4-(2,4-dimethoxy-3-methylphenethyl)benzene-1,3-diol (19) as described inExample 7. The data is presented as melanin content (% of untreatedcontrol) vs. inhibitor concentration (μM). The IC₅₀ of compound 19 wasdetermined to be 24.7 μM. FIG. 6 also illustrates graphically cellviability (LD₅₀) (x) at varying concentrations of compound 19. The LD₅₀for compound 19 was determined to be 130.3 μM.

FIG. 7 illustrates graphically a profile of the inhibition in theproduction of melanin (●) by 4-(2-(furan-2-yl)ethyl)benzene-1,3-diol(24) as described in Example 7. The data is presented as melanin content(% of untreated control) vs. inhibitor concentration (μM). The IC₅₀ ofcompound 24 was determined to be 1.6 μM. FIG. 7 also illustratesgraphically cell viability (LD₅₀) (x) at varying concentrations ofcompound 24. The LD₅₀ for compound 24 was determined to be 345.9 μM.

FIG. 8 illustrates graphically a profile of the inhibition in theproduction of melanin (●) by4-(2-(tetrahydrofuran-2-yl)ethyl)benzene-1,3-diol (25) as described inExample 7. The data is presented as melanin content (% of untreatedcontrol) vs. inhibitor concentration (μM). The IC₅₀ of compound 25 wasdetermined to be 5 μM. FIG. 8 also illustrates graphically cellviability (LD₅₀) (x) at varying concentrations of compound 25. The LD₅₀for compound 25 was determined to be >1000 μM.

FIG. 9 illustrates graphically a profile of the inhibition in theproduction of melanin (●) by compound 30 as described in Example 7. Thedata is presented as melanin content (% of untreated control) vs.inhibitor concentration (μM). The IC₅₀ of compound 30 was determined tobe 156 μM. FIG. 9 also illustrates graphically cell viability (LD 50)(x) at varying concentrations of compound 25. The LD₅₀ for compound 30was determined to be 187.8 μM.

FIG. 10 illustrates graphically a profile of the inhibition in theproduction of melanin (●) by kojic acid as described in Example 7. Thedata is presented as melanin content (% of untreated control) vs.inhibitor concentration (μM). The IC₅₀ of kojic acid in the murine assaywas determined to be 303.5 μM. FIG. 10 also illustrates graphically cellviability (LD₅₀) (x) at varying concentrations of kojic acid. The LD₅₀for kojic acid was determined to be >1000 μM.

FIG. 11A depicts the reconstructed skin prepared as described in Example8. The reconstructed skin was grown at the air-liquid interface, makingit possible to mimic topical application of skin whitening agents.

FIG. 11B depicts photographically the results of the reconstructed skinstudies as described in Example 8. Photographs of skin specimen takenafter 15 days of the experiment show significant dose-dependentwhitening effects on melanocytes, which appear on photographs as darkdendritic cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present disclosure provides pharmaceutical agents that are potentinhibitors of the tyrosinase enzyme. In particular, provided aresubstituted 2,4-dihydroxybenzene derivatives that are inhibitors oftyrosinase having the structures depicted in Formulas I-IV, below orpharmaceutically acceptable salts thereof.

These compounds are shown to have excellent tyrosinase inhibitoryactivity and low cytotoxicity. Illustrated is the inhibitory activity ofrepresentative compounds against mushroom tyrosinase, as well as, theinhibition of melanin production by murine B16-F1 melanoma cells. Theskin whitening properties of representative compounds were evaluated bymeans of a reconstructed human skin model as described in Example 8.These compounds or their pharmaceutically acceptable salts thereof areshown to be useful for both cosmetic and medical applications asdetailed below.

As used in this context, the term “derivative” or “analog” refers to acompound having similar chemical structure or function as the compoundsof Formula I-IV that retains the core 2,4-dihydroxybenzene ring.

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.Unless defined otherwise all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs.

It is to be noted that as used herein the term “a” or “an” entity refersto one or more of that entity; for example, a tyrosinase inhibitorrefers to one or more tyrosinase inhibitors. As such, the terms “a” or“an”, “one or more” and “at least one” are used interchangeably herein.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term.

The term “alkyl” as used herein refers to a straight or branched chain,saturated hydrocarbon having the indicated number of carbon atoms. Forexample, (C₁-C₁₀) alkyl is meant to include a straight or branched chainhydrocarbon having one to ten carbon atoms. An alkyl group can beunsubstituted or optionally substituted with one or more substituents asdescribed herein.

The term “alkoxy” as used herein refers to an —O-alkyl group having theindicated number of carbon atoms. For example, a (C₁-C₁₀) alkoxy groupincludes —OCH₃, —OCH₂CH₃, etc. up to ten carbon atoms.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N) and sulfur (S).

The term “heteroaromatic” as used herein refers to an aromaticheterocyclic ring of 5 to 14 members and having at least one heteroatomselected from nitrogen, oxygen and sulfur, and containing at least 1carbon atom. Representative heteroaromatics include pyridyl, furyl,thienyl, pyrrolyl and imidazolyl etc. The heteroaryl group can beattached via any heteroatom or carbon atom, where chemically acceptable.A heteroaryl group can be unsubstituted or optionally substituted withone or more substituents as described herein.

As used herein, the term “heterocycle” refers to non-aromatic 5 to14-membered ring systems which are either saturated, unsaturated andwhich contain from 1 to 4 heteroatoms independently selected fromnitrogen, oxygen and sulfur, and wherein the nitrogen and sulfurheteroatoms can be optionally oxidized, and the nitrogen heteroatom canbe optionally quaternized. The heterocycle can be attached via anyheteroatom or carbon atom, where chemically acceptable. Representativeexamples of non-aromatic heterocycles include, but are not limited totetrahydrofuranyl, tetrahydropyrrolyl, pyranyl and tetrahydropyranyletc. A heterocycle group can be unsubstituted or optionally substitutedwith one or more substituents as described herein.

The term “aromatic” as used herein refers to any compound which includesor consists of one or more hydrocarbon aromatic rings. The rings may bemono or polycyclic ring systems. Examples of suitable rings include, butare not limited to benzene, biphenyl, terphenyl, naphthalene etc.

The term “hydroxyalkyl” as used herein, refers to an alkyl group havingthe indicated number of carbon atoms wherein one or more of the hydrogenatoms in the alkyl group is replaced with an —OH group. Representativeexamples of hydroxyalkyl groups include, but are not limited to, —CH₂OH,—CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂CH₂OH, andbranched versions thereof. Typically, the hydroxylalkyl is a compound ofthe formula —(C₁-C₁₀)alkyl-OH.

“Therapeutic” as used herein, includes prevention, treatment and/orprophylaxis. When used therapeutic refers to humans as well as otheranimals.

“Pharmaceutically or therapeutically effective dose or amount” refers toa dosage level sufficient to induce a desired biological result. Thatresult may be the alleviation of the signs, symptoms or causes of adisease or any other alteration of a biological system that is desired.The precise dosage will vary according to a variety of factors,including but not limited to the age and size of the subject, thedisease and the treatment being effected.

A “host” or “patient” or “subject” is a living mammal, human or animal,for whom therapy is desired. The “host,” “patient” or “subject”generally refers to the recipient of the therapy to be practicedaccording to the method of the invention. It should be noted that theinvention described herein may be used for veterinary as well as humanapplications and that the term “host” should not be construed in alimiting manner. In the case of veterinary applications, the dosageranges can be determined as described below, taking into account thebody weight of the animal.

As used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of a federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals and, more particularly, in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which the therapeuticis administered and includes, but is not limited to such sterile liquidsas water and oils.

A “pharmaceutically acceptable salt” or “salt” of a tyrosinase inhibitoris a product of the disclosed compound that contains an ionic bond, andis typically produced by reacting the disclosed compound with either anacid or a base, suitable for administering to a subject. Apharmaceutically acceptable salt can include, but is not limited to,acid addition salts including hydrochlorides, hydrobromides, phosphates,sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates,arylalkylsulfonates, acetates, benzoates, citrates, maleates, fumarates,succinates, lactates, and tartrates; alkali metal cations such as Li,Na, K, alkali earth metal salts such as Mg or Ca, or organic aminesalts.

A “pharmaceutical composition” is a formulation comprising the disclosedcompounds in a form suitable for administration to a subject. Apharmaceutical composition of the invention is preferably formulated tobe compatible with its intended route of administration. Examples ofroutes of administration include, but are not limited to, oral andparenteral, e.g., intravenous, intradermal, subcutaneous, inhalation,topical, transdermal, transmucosal, and rectal administration.

The term “substituted,” as used herein, means that any one or morehydrogens on the designated atom is replaced with a selection from theindicated group, provided that the designated atom's normal valency isnot exceeded, and that the substitution results in a stable compound.When a substituent is keto (i.e., ═O), then 2 hydrogens on the atom arereplaced. Examples of substituents include, but are not limited toC₁-C₁₀ alkyl, hydroxy (—OH); C₁-C₁₀ alkoxy groups. Typically anaromatic, heteroaromatic or heterocyclic ring will have from 1-3substituents.

In one of its aspects the present invention provides a compound having astructure shown in Formula I, or a pharmaceutically acceptable saltthereof:

wherein R is selected from a substituted or unsubstituted aromatic,heteroaromatic or heterocyclic ring. In one embodiment R is selectedfrom the group consisting of a substituted or unsubstituted: phenyl,biphenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furanyl, 3-furanyl. In oneembodiment R is substituted with 1 to 3 moieties (R′, R″, R′″)independently selected from the group consisting of a C₁-C₁₀ alkylgroup, a C₁-C₁₀ alkoxy group or a hydroxyl group. In other embodiments Ris selected from the group consisting of 1′-(4′-methoxy)phenyl;1′-(2,4-dihydroxy)phenyl; 1′-(3-pyridyl); 1′-(biphenyl-4-ol).

In one of its aspects the present invention provides a compound having astructure shown in Formula II or a pharmaceutically acceptable saltthereof.

wherein X═H or —OH and R is selected from a substituted or unsubstitutedaromatic, heteroaromatic or heterocyclic ring. In one embodiment R isselected from the group consisting of a substituted or unsubstituted:phenyl, biphenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furanyl, 3-furanyl.In one embodiment, R is substituted with 1 to 3 moieties (R′, R″, R′″)independently selected from the group consisting of a C₁-C₁₀ alkylgroup, a C₁-C₁₀ alkoxy group or a hydroxyl group. In other embodiments Ris selected from the group consisting of 2′-furyl; 3′-furyl; 3′-pyridyl;3′-(2-methoxypyridyl); 1′-(2,4,6-trimethoxy phenyl); 1′-(3,4-dioxalanephenyl) and when X═OH, R is 3′-pyridyl.

In one of its aspects the present invention provides a compound having astructure shown in Formula III or a pharmaceutically acceptable saltthereof.

wherein R₁ is selected from the group consisting of H, or C₁-C₁₀ alkyl;and R₂ is selected from a substituted or unsubstituted: aromatic ring,heteroaromatic ring or heterocyclic ring. In particular embodiments R₁is selected from H or —CH₃ and R₂ is selected from a substituted orunsubstituted: aromatic ring, heteroaromatic ring or heterocyclic ring.In yet other embodiments, R₂ is substituted with 1 to 3 moieties (R′,R″, R′″) independently selected from the group consisting of a C₁-C₁₀alkyl group, a C₁-C₁₀ alkoxy group or a hydroxyl group. In otherembodiments R₂ is selected from the group consisting of a substituted orunsubstituted: phenyl, biphenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-furanyl, 3-furanyl or 2-tetrahydrofuranyl. In specific embodiments R₂is selected from the group consisting of R₁ is H and R₂ is 1′-phenyl;1′-(4-hydroxyphenyl); 1′-(4-methoxyphenyl); 1′-(3,4-dioxalane)phenyl;1′-(2,4-dihydroxyphenyl); 1′-(2,4-dimethoxyphenyl);1′-(3,5-dimethoxyphenyl); 1′-(2,4-dimethoxy-3-methylphenyl);1′-(2,4,6-trimethoxyphenyl); 3-furyl; 3-tetrahydrofuryl; 2′-furyl,2′-tetrahydrofuryl; 2′-(5′-ethyl)-furyl; 3′-pyridyl and also R¹=Me,R₂=1′-phenyl

In one of its embodiments the present invention provides a compoundhaving a structure shown in Formula II or a pharmaceutically acceptablesalt thereof.

wherein

represents double or single bond,

-   -   R₁ is selected from the group consisting of H or C₁-C₁₀ alkyl;    -   R₄ is selected from the group consisting of ═H, OH or O; and    -   R₅ is a selected from the group consisting of a substituted or        unsubstituted: aromatic ring, heteroaromatic ring, heterocyclic        ring or a hydroxylalkyl moiety. In specific embodiments R₁ is H        or CH₃ and R₅ is selected from a substituted or unsubstituted:        aromatic ring, heteroaromatic ring or a heterocyclic ring or a        C₁-C₁₀ hydroxylalkyl (—C₁-C₁₀)—OH). In yet other embodiments, R₅        is substituted with 1 to 3 moieties (R′, R″, R′″) independently        selected from the group consisting of a C₁-C₁₀ alkyl group, a        C₁-C₁₀ alkoxy group or a hydroxyl group. In other embodiments R₅        is selected from the group consisting of a substituted or        unsubstituted: phenyl, biphenyl, 2-pyridyl, 3-pyridyl,        4-pyridyl, 2-furanyl, 3-furanyl or 2-tetrahydrofuranyl. In        specific embodiments R₁═R₄═H and R₅ is selected from        1′-(3′,5′-dimethoxy)phenyl; 1′-(3′,5′-dihydroxy)phenyl;        1-(2′,4′,6-trimethoxyphenyl); R₁=Me, R₄═OH, R₅ is        1′-(2′,4′-dihydroxy)phenyl; R₁=Me, R₄=0, R₅ is        1′-(2′,4′-dihydroxy)phenyl; and R₁═R₄═H, R₅—CH₂CH₂OH.    -   with the following provisos:    -   proviso 1: when R₁═R₄═H; R₅ cannot be        1′-(2,4-dimethoxy-3-methyl)phenyl. proviso 2: when R₁═R₄═H; R₅        cannot be 1′-(2,4-dihydroxy)phenyl; 1′-(2,4-dialkoxy)phenyl;        1′-(2,4-dialkenoxy)phenyl or 1′-(2,4-dibenzyloxy)phenyl.    -   proviso 3: R₅ cannot be 1′-(4-hydroxy)phenyl.    -   proviso 4: when R₁═R₄═H; R₅ cannot be 1′-(2,4-di(CO)R¹, wherein        R¹ is a (C₁-C₂₀)-alkyl group.    -   proviso 5: when R₁═R₄═H and R₅ cannot be a        1′-(2,4-disubstituted)phenyl; wherein said substituents are        selected from —OR² and —OR³, wherein R₂ and R₃ are independently        selected from the group consisting of H or a (C₁-C₂₀)-alkyl.    -   proviso 6: when R₁═H and R₄═OH; R₅ cannot be        1′-(3,4-dihydroxy)phenyl; 1′-(3,4-dimethoxy)phenyl;        1′-(2-hydroxy)phenyl; 1′-(2-methoxy)phenyl;        1′-(4-methoxy)phenyl; phenyl; 1′-(3-methoxy,4-hydroxy)phenyl.

Table 1 lists representative novel 2,4-dihydroxybenzene analogs ofFormula I which are useful as tyrosinase inhibitors. The syntheticmethods that can be used to prepare each compound, identified in Table 1are described in detail in Example 1. Supporting ¹H- and ¹³C-NMR data isprovided for each compound synthesized. In general, the compounds ofFormula I can be synthesized from readily available materials usingstandard organic synthesis techniques. Further preparation routes may befound in the literature and relevant art. IC₅₀ values for thesecompounds as determined by the tyrosinase assay as described in Example6 are also set forth in Table 1. For select compounds of Formula I,Table 5 provides IC₅₀ values as determined by the murine melanomacell-based assays described in Example 7 and cell viability (LD₅₀) asdetermined by the methods described in Example 8.

Table 2 lists representative novel 2,4-dihydroxybenzene derivatives ofFormula II which are useful as tyrosinase inhibitors. The syntheticmethods that can be used to prepare each compound identified in Table 2are described in detail in Example 2. Supporting ¹H- and ¹³C-NMR data isprovided for each compound synthesized. In general, the compounds ofFormula II can be synthesized from readily available materials usingstandard organic synthesis techniques. Further preparation routes may befound in the literature and relevant art. IC₅₀ values for thesecompounds as determined by the tyrosinase assay as described in Example6 are also set forth in Table 2. For select compounds of Formula II,Table 5 provides IC₅₀ values as determined by the murine melanomacell-based assay described in Example 7 and cell viability (LD₅₀) asdetermined by the methods described in Example 8.

Table 3 lists representative novel 2,4-dihydroxybenzene analogs ofFormula III which are useful as tyrosinase inhibitors. The syntheticmethods that can be used to prepare each compound identified in Table 3are described in detail in Example 3. Supporting ¹H- and ¹³C-NMR data isprovided for each compound synthesized. In general, the compounds ofFormula III can be synthesized from readily available materials usingstandard organic synthesis techniques. Further preparation routes may befound in the literature and relevant art. IC₅₀ values for thesecompounds as determined by the tyrosinase assay as described in Example6 are also set forth in Table 3. For select compounds of Formula III,Table 5 provides IC₅₀ values as determined by the murine melanomacell-based assay described in Example 7 and cell viability (LD₅₀) asdetermined by the methods described in Example 8.

Table 4 lists representative novel 2,4-dihydroxybenzene analogs ofFormula IV which are useful as tyrosinase inhibitors. The syntheticmethods that can be used to prepare each compound identified in Table 4are described in detail in Example 4 and Example 5 (compound 34).Supporting ¹H- and ¹³C-NMR data is provided for each compoundsynthesized. In general, the compounds of Formula IV can be synthesizedfrom readily available materials using standard organic synthesistechniques. Further preparation routes may be found in the literatureand relevant art. IC₅₀ values for these compounds as determined by thetyrosinase assay as described in Example 6 are also set forth in Table4. For select compounds of Formula IV, Table 5 provides IC₅₀ values asdetermined by the murine melanoma cell-based assay described in Example7 and cell viability (LD₅₀) as determined by the methods described inExample 8.

In summary, the compounds set forth shown in Tables 1-4 exhibitedexcellent inhibition of the mushroom tyrosinase enzyme, as well as,melanin production by B16-F1 cells (Table 5, FIGS. 1-9 ). Cytotoxicitywas observed only at high concentrations. All compounds exemplified inTable 5 (i.e., compounds 7, 15, 16, 17, 18, 19, 24, 25, 30) were muchmore potent than kojic acid, a commonly used skin whitener asillustrated in FIG. 10 . The IC₅₀ of kojic acid (tyrosinase assay) is 20μM. The IC₅₀ of kojic acid (murine assay) is 303.5 μM. Additionally, twoof the compounds tested (compounds 16 and 25) exhibited strongskin-whitening effects in a reconstructed skin model without anydetectable cytotoxicity as described in Example 8.

The present invention encompasses pharmaceutical compositions comprisingat least one tyrosinase inhibitor described herein. The compositions ofthe present invention can be formulated as pharmaceutical compositions,which include other components such as a pharmaceutically and/orcosmetically acceptable excipient, an adjuvant, and/or a carrier. Forexample, compositions of the present invention can be formulated in anexcipient that the host to be treated can tolerate. An excipient is aninert substance used as a diluent or vehicle for a therapeutic agent.Examples of such excipients include, but are not limited to water,buffers, saline, Ringer's solution, dextrose solution, mannitol, Hank'ssolution, preservatives and other aqueous physiologically balanced saltsolutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyloleate, or triglycerides may also be used. Other useful formulationsinclude suspensions containing viscosity-enhancing agents, such assodium carboxymethylcellulose, sorbitol or dextran. Excipients can alsocontain minor amounts of additives, such as substances that enhanceisotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer, tris buffer, histidine, citrate,and glycine, or mixtures thereof, while examples of preservativesinclude, but are not limited to EDTA, disodium EDTA, BHA, BHT, vitaminC, vitamin E, sodium bisulfite, SnCl₂, thimerosal, m- or o-cresol,formalin and benzyl alcohol. Standard formulations can be either liquidor solids, which can be taken up in a suitable liquid as a suspension orsolution for administration. Thus, in a non-liquid formulation, theexcipient can comprise dextrose, human serum albumin, preservatives,etc., to which sterile water or saline can be added prior toadministration.

In one embodiment of the present invention, the composition can alsoinclude an adjuvant or a carrier. Adjuvants are typically substancesthat generally enhance the biological response of a host to a specificbioactive agent. Suitable adjuvants include, but are not limited to,Freund's adjuvant, other bacterial cell wall components, aluminum,magnesium, copper, zinc, iron, calcium, and other metal ion based salts,silica, polynucleotides, toxoids, serum proteins, viral coat proteins,other bacterial-derived preparations, gamma interferon; block copolymeradjuvants; such as Hunter's Titermax adjuvant (Vaxcel™, Inc. Norcross,Ga.), Ribi adjuvants (available from Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives, such as Quil A(available from Superfos Biosector A/S, Denmark). Carriers are typicallycompounds that increase the half-life of a therapeutic composition inthe treated host. Suitable carriers include, but are not limited to,polymeric controlled release formulations, biodegradable implants,liposomes, bacteria, viruses, oils, esters, and glycols. Suitablecarriers are described in “Remington: The Science and Practice,Twentieth Edition,” published by Lippincott Williams & Wilkins, which isincorporated herein by reference.

In one embodiment, the composition is prepared as a controlled releaseformulation, which slowly releases the composition of the presentinvention into the host. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehicles will beknown to those skilled in the art. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

The compositions of this invention can be administered by any methodknown to one of ordinary skill in the art. The modes of administrationinclude, but are not limited to, enteral (oral) administration,parenteral (intravenous, subcutaneous, and intramuscular) administrationand topical application. The method of treatment according to thisinvention comprises administering internally or topically to a patientin need thereof a therapeutically effective amount of at least one ofthe 2,4-dihydroxybenzene analogs of the instant invention.

In one embodiment, the therapeutic agents of the instant invention areadministered topically by any suitable means, known to those of skill inthe art for topically administering therapeutic compositions including,but not limited to as an ointment, gel, lotion, or cream base, or as atoothpaste, mouth-wash, or coated on dental flossing materials or as anemulsion, as a patch, dressing or mask, a nonsticking gauze, a bandage,a swab or a cloth wipe.

A therapeutic composition can be administered in a variety of unitdosage forms depending upon the method of administration. For particularmodes of delivery, a therapeutic composition of the present inventioncan be formulated in an excipient of the present invention. Atherapeutic reagent of the present invention can be administered to anyhost, preferably to mammals, and more preferably to humans. Theparticular mode of administration will depend on the condition to betreated.

In one embodiment, a suitable ointment is comprised of the desiredconcentration of at least one 2,4-dihydroxybenzene analog of the instantinvention, that is an efficacious, nontoxic quantity generally selectedfrom the range of 0.001% to 100% based on the total weight of thetopical formulation, from 65 to 100% (preferably 75 to 96%) of whitesoft paraffin, from 0 to 15% of liquid paraffin, and from 0 to 7%(preferably 3 to 7%) of lanolin or a derivative or synthetic equivalentthereof. In another embodiment the ointment may comprise apolyethylene-liquid paraffin matrix.

In one embodiment, a suitable cream is comprised of an emulsifyingsystem together with the desired concentration of at least one2,4-dihydroxybenzene analog of the instant invention as provided above.The emulsifying system is preferably comprised of from 2 to 10% ofpolyoxyethylene alcohols (e.g. the mixture available under the trademarkCetomacrogol™ 1000), from 10 to 25% of stearyl alcohol, from 20 to 60%of liquid paraffin, and from 10 to 65% of water; together with one ormore preservatives, for example from 0.1 to 1% ofN,N″-methylenebis[N′-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea](available under the name Imidurea USNF), from 0.1 to 1% of alkyl4-hydroxybenzoates (for example the mixture available from NipaLaboratories under the trade mark Nipastat), from 0.01 to 0.1% of sodiumbutyl 4-hydroxybenzoate (available from Nipa Laboratories under thetrade mark Nipabutyl sodium), and from 0.1 to 2% of phenoxyethanol.

In one embodiment, a suitable gel is comprised of a semi-solid system inwhich a liquid phase is constrained within a three dimensional polymericmatrix with a high degree of cross-linking. The liquid phase may becomprised of water, together with the desired amount of at least one2,4-dihydroxybenzene analog of the instant invention, from 0 to 20% ofwater-miscible additives, for example glycerol, polyethylene glycol, orpropylene glycol, and from 0.1 to 10%, preferably from 0.5 to 2%, of athickening agent, which may be a natural product, selected from thegroup including, but not limited to tragacanth, pectin, carrageen, agarand alginic acid, or a synthetic or semi-synthetic compound, selectedfrom the group including, but not limited to methylcellulose andcarboxypolymethylene (carbopol); together with one or morepreservatives, selected from the group including, but not limited to forexample from 0.1 to 2% of methyl 4-hydroxybenzoate (methyl paraben) orphenoxyethanol-differential. Another suitable base, is comprised of thedesired amount of at least one 2,4-dihydroxybenzene analog of theinstant invention, together with from 70 to 90% of polyethylene glycol(for example, polyethylene glycol ointment containing 40% ofpolyethylene glycol 3350 and 60% of polyethylene glycol 400, prepared inaccordance with the U.S. National Formulary (USNF)), from 5 to 20% ofwater, from 0.02 to 0.25% of an anti-oxidant (for example butylatedhydroxytoluene), and from 0.005 to 0.1% of a chelating agent (forexample ethylenediamine tetraacetic acid (EDTA)).

The term soft paraffin as used above encompasses the cream or ointmentbases white soft paraffin and yellow soft paraffin. The term lanolinencompasses native wool fat and purified wool fat. Derivatives oflanolin include in particular lanolins which have been chemicallymodified in order to alter their physical or chemical properties andsynthetic equivalents of lanolin include in particular synthetic orsemisynthetic compounds and mixtures which are known and used in thepharmaceutical and cosmetic arts as alternatives to lanolin and may, forexample, be referred to as lanolin substitutes.

One suitable synthetic equivalent of lanolin that may be used is thematerial available under the trademark Softisan™ known as Softisan 649.Softisan 649, available from Dynamit Nobel Aktiengesellschaft, is aglycerine ester of natural vegetable fatty acids, of isostearic acid andof adipic acid; its properties are discussed by H. Hermsdorf in Fette,Seifen, Anstrichmittel, Issue No. 84, No. 3 (1982), pp. 3-6.

The other substances mentioned hereinabove as constituents of suitableointment or cream bases and their properties are discussed in standardreference works, for example pharmacopoeia. Cetomacrogol 1000 has theformula CH₃(CH₂)_(m)(OCH₂CH₂)_(n)OH, wherein m may be 15 or 17 and n maybe 20 to 24. Butylated hydroxytoluene is 2,6-di-tert-butyl-p-cresol.Nipastat is a mixture of methyl, ethyl, propyl and butyl4-hydroxybenzoates.

The compositions of the invention may be produced by conventionalpharmaceutical techniques. Thus the aforementioned compositions, forexample, may conveniently be prepared by mixing together at an elevatedtemperature, preferably 60-70° C., the soft paraffin, liquid paraffin ifpresent, and lanolin or derivative or synthetic equivalent thereof. Themixture may then be cooled to room temperature, and, after addition ofthe hydrated crystalline calcium salt of mupirocin, together with thecorticosteroid and any other ingredients, stirred to ensure adequatedispersion.

Regardless of the manner of administration, the specific dose iscalculated according to the approximate body weight of the host. Furtherrefinement of the calculations necessary to determine the appropriatedosage for treatment involving each of the above mentioned formulationsis routinely made by those of ordinary skill in the art and is withinthe scope of tasks routinely performed by them without undueexperimentation, especially in light of the dosage information andassays disclosed herein. These dosages may be ascertained through use ofthe established assays for determining dosages utilized in conjunctionwith appropriate dose-response data.

The tyrosinase inhibitors of the invention can readily be synthesizedusing known synthetic methodologies or via a modification of knownsynthetic methodologies. As would be readily recognized by a skilledartisan, the methodologies described below allow the synthesis of2,4-dihydroxybenzene analogs having a variety of substituents. Includedin this invention is a method of synthesizing the 2,4-dihydroxybenzenanalogs described herein. Exemplary synthetic methods are described inExamples 1-5 below.

The invention encompasses methods of preventing or treating (e.g.,alleviating one or more symptoms of) medical conditions through use ofone or more of the disclosed compounds. The methods of preventing ortreating comprise administering a therapeutically effective amount of atleast one tyrosinase inhibitor of the instant invention to a patient inneed thereof. The compositions of the invention can also be used forprophylactic therapy.

The present invention provides a method of treating a subject afflictedwith a disorder ameliorated by the inhibition of the activity tyrosinaseor the production of excess amounts of melanin. Such a method comprisesadministering to a subject a therapeutically effective amount of atyrosinase inhibitor as described herein.

As used herein, “treating” describes the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes the administration of a compound of the present inventionto prevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition ordisorder. More specifically, “treating” includes reversing, alleviating,minimizing, suppressing or halting at least one deleterious symptom oreffect of a disease (disorder) state, disease progression, or otherabnormal condition. Treatment is continued as long as symptoms and/orpathology ameliorate.

More specifically the present invention also provides a method forinhibiting the synthesis of melanin in a subject in need thereof. Such amethod comprises administering an effective amount of a compositioncomprising at least one of the 2,4-dihydroxybenzene analogs of theinstant invention.

The present invention further provides methods for the prevention andtreatment of diseases and conditions related to the overproduction oruneven distribution of melanin, said method comprising administeringinternally or topically to a subject in need thereof a therapeuticallyeffective amount of at least one at least one 2,4-dihydroxybenzenecompound of the instant invention. Diseases and conditions related tothe overproduction or uneven distribution of melanin include, but notlimited to hyperpigmentation spots caused by skin aging, melasma, liverdiseases, thermal burns and topical wounds, skin pigmentation due toinflammatory conditions caused by fungal, microbial and viralinfections, vitilago, carcinoma, melanoma, as well as other mammalianskin conditions.

The method can also be used for preventing and treating skin darkeningand damage resulting from exposure to the sun, ultraviolet (UV)radiation, chemicals, heat, wind and dry environments. Finally, themethod can be used for preventing and treating wrinkles, saggy skin,lines and dark circles around the eyes, soothing sensitive skin andpreventing and treating dermatitis and other allergy related conditionsof the skin.

In addition to their use for the prevention and treatment of the abovedescribed diseases and conditions of the skin, the therapeuticcompositions described herein provide an efficacious composition thatyields the benefit of smooth and youthful skin appearance with improvedskin color, enhanced elasticity, reduced and delayed aging, enhancedyouthful appearance and texture, and increased flexibility, firmness,smoothness and suppleness.

In yet another embodiment, the 2,4-dihydroxybenzene derivatives of theinstant invention can be used in the food industry to inhibit thebrowning and color changes in fruits, vegetables, juices and other foodproducts.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including a U.S. patent, are specificallyincorporated by reference.

EXAMPLES Example 1. General and Specific Methods for the Synthesis ofCompounds of Formula I

Compounds of Formula I were prepared generally as set forth in Scheme 1,using a substituted bromobenzene as R for purposes of illustration.Briefly, the biphenyl compounds of Formula I were prepared by Kumadacoupling reaction (Tamao, K.; Sumitani, K.; Kumada, M, Journal of theAmerican Chemical Society. 1972, 94. 4374-4376) using Ni(dppp)Cl₂catalyst from the corresponding Grignard reagents with appropriate arylbromide followed by reductive debenzylation reaction of benzyl group.

Representative Procedure for Scheme 1: Synthesis of4′-Methoxybiphenyl-2,4-diol (1)

4-Bromoanisole (2.80 g, 15 mmol) was treated with magnesium turnings(480 mg, 20 mmol) and iodine (a pinch) in THE to make the correspondingGrignard reagent. The Grignard reagent was then added to a stirredsolution of benzyl protected 4-bromoresorcinol (3.7 g, 10 mmol) andNi(dppp)Cl₂ (2.7 g, 5 mmol) in THE at 0° C. The reaction mixture wasallowed to stir at room temperature for 30 minutes after which it washeated to reflux for 4 h. The reaction mixture was then cooled down andquenched with ammonium chloride, extracted with ethyl acetate, washedwith brine and dried over anhydrous Na₂SO₄, filtered and concentrated inrotary evaporator and purified by column chromatography to yield (1.98g, 50%) of the coupled product as an off white solid.

The resulting coupled product (1.5 g, 3.7 mmol) was then treated with acatalytic amount of 10% Pd on activated carbon (150 mg) and an excessamount of ammonium formate (1.9 g, 29.6 mmol) in 15 mL THF/MeOH (1:2)solution at reflux for 3 h to remove the benzyl protection group. Afterfiltration through Celite to remove the catalyst, the crude product waspurified by column chromatography to yield (720 mg, 90%) of compound 1as an off-white solid. ¹H-NMR (MeOD, 500 MHz): δ 7.152 (d, 2H, J=8.5Hz), 6.718-6.762 (m, 3H), 6.443 (d, 1H, J=2.5 Hz), 6.241 (dd, 1H, J=2.5& 8.0 Hz), 3.663 (s, 3H).

Representative compounds 2-4 of Formula I (see Table 1) were synthesizedaccording to the same protocol using appropriate staring materials.Overall yields varied from 40-50%.

Biphenyl-2,2′,4,4′-tetraol (2)

¹H-NMR (MeOD, 300 MHz): δ 7.007-6.953 (m, 2H), 6.378-6.327 (m, 6H).¹³C-NMR (MeOD, 75 MHz): δ 157.971 (6C), 130.246 (2CH), 107.134 (2CH),102.589 (2CH).

4-(Pyridin-3yl)benzene-1,3-diol (3)

¹H-NMR: (CDCl₃, 300 MHz): δ 8.829 (s, 1H), 8.539 (d, 1H, J=3.6 Hz),7.885 (d, 1H, J=7.5 Hz), 7.605-7.261 (m, 1H), 6.753 (s, 1H), 6.711 (d,1H, J=8.4 Hz). ¹³C-NMR (CDCl₃, 75 MHz): δ 157.036 (C), 150.534 (CH),137.420 (CH), 136.926 (CH), 134.400 (C), 131.618 (CH), 129.237 (C),127.931 (CH), 120.872 (C), 106.821 (CH), 101.769 (CH).

4-(4′-Hydroxybiphenyl)benzene-1,3-diol (4)

¹H-NMR (MeOD, 300 MHz): δ 7.511-7.183 (m, 9H), 6.88-6.83 (m, 2H).¹³C-NMR (MeOD, 75 MHz): δ 156.949 (3 C), 141.204 (2 C), 132.709 (2 C),128.557 (2 CH), 127.893 (3 CH), 126.256 (3 CH), 115.472 (3 CH).

TABLE 1 Compounds Representative of Formula I Mushroom CompoundStructure Tyrosinase Assay 4′-Methoxybiphenyl-2,4-diol (1)

IC₅₀: = 12.8 μM Biphenyl-2,2′,4,4′-tetraol (2)

IC₅₀: = 60.0 μM 4-(Pyridin-3yl)benzene-1,3-diol (3)

IC₅₀: = 0.5 μM 4-(4′-Hydroxybiphenyl)benzene- 1,3-diol (4)

IC₅₀: = 70.0 μM

Example 2. General and Specific Methods for the Synthesis of Compoundsof Formula II

Compounds of Formula II were prepared as illustrated in Scheme 2.Briefly, with reference to Scheme 2, the diphenylmethane compoundsillustrative of Formula II were prepared by Grignard addition reactionwith an aldehyde (RCHO, wherein R=aromatic or heteroaromatic ring),followed by dehydroxylation of resulting benzylic hydroxyl group anddebenzylation of benzyloxy protecting groups in situ.

Representative Procedure for Scheme 2: Synthesis of4-(Furan-2-ylmethyl)benzene-1,3-diol (5)

Benzyl protected 4-bromoresorcinol (2.80 g, 15 mmol) was treated withmagnesium turnings (480 mg, 20 mmol) and iodine (a pinch) in THE toprovide the corresponding Grignard reagent, which was then added to astirred solution of 2-furaldehyde (3.97 g, 10 mmol) in THE at 0° C. Thereaction mixture was allowed to warm up to room temperature and stirredat room temperature for 3 h. The mixture was then quenched with ammoniumchloride, extracted with ethyl acetate, washed with brine and dried overanhydrous Na₂SO₄, filtered, concentrated in a rotary evaporator andpurified by column chromatography to provide (3.0 g, 80%) of the coupledproduct.

The coupled product (2.5 g, 6.5 mmol) was then treated with a catalyticamount of 10% Pd on activated carbon (250 mg) and an excess amount ofammonium formate (3.33 g, 52 mmol) in THF/MeOH (18 mL) at reflux toremove the benzyl protecting groups. Partial dehydroxylation of benzylhydroxy group also took place during the debenzylation reaction. Uponcompletion, the reaction mixture was filtered through Celite to removethe catalyst and concentrated to provide the crude product, which wasthen purified by column chromatography to yield (740 mg, 60%) ofcompound 5 as an off-white solid, as well as, the hydroxylated compound(X═OH) (270 mg, 20%). ¹H-NMR (MeOD, 500 MHz): δ 7.306 (dd, 1H, J=1.0 &2.0 Hz), 6.8565 (d, 1H, J=8.5 Hz), 6.8085 (d, 1H, J=8.5 Hz), 6.3155 (d,1H, J=2.5 Hz), 6.2965 (d, 1H, J=2.5 Hz), 6.255-6.223 (m, 2H). ¹³C-NMR(MeOD, 125 MHz): δ 156.604 (C), 156.504 (C), 156.064 (C), 155.556 (C),155.290 (C), 140.575 (CH), 131.443 (CH), 130.325 (CH), 116.098 (C),116.973 (C), 109.761 (CH), 106.303 (CH), 106.111 (CH), 105.156 (CH),102.513 (CH), 102.061 (CH), 79.946 (CH), 67.489 (CH₂), 35.257 (CH),29.875 (CH₂), 27.102 (CH₂), 25.218 (CH₂).

Representative compounds 6-11 of Formula II (see Table 2) weresynthesized according to the same general reaction scheme using theappropriate staring materials. Overall yields varied from 50-60%.

4-(Furan-3-ylmethyl)benzene-1,3-diol (6)

¹H-NMR (MeOD, 500 MHz): δ 8.39 (d, 1H, J=2 Hz), 8.28 (dd, 1H, J=5 & 1.5Hz), 6.63-6.69 (m, 1H), 7.25-7.28 (m, 1H), 6.87 (d, 1H, J=8.5 Hz), 6.31(d, 1H, J=2.5 Hz), 6.24 (dd, 1H, J=8 & 2.5 Hz), 3.25 (s, 2H). ¹³C-NMR(MeOD, 125 MHz): δ 156.897 (C), 155.797 (C), 148.727 (CH), 145.584 (CH),138.672 (C), 137.021 (CH), 130.536 (CH), 123.476 (CH), 117.446 (C),106.221 (CH), 102.283 (CH), 32.196 (CH₂).

4-(Pyridin-ylmethyl)benzene-1,3-diol (7)

¹H-NMR (MeOD, 500 MHz): δ 8.39 (d, 1H, J=2 Hz), 8.28 (dd, 1H, J=5 & 1.5Hz), 6.63-6.69 (m, 1H), 7.25-7.28 (m, 1H), 6.87 (d, 1H, J=8.5 Hz), 6.31(d, 1H, J=2.5 Hz), 6.24 (dd, 1H, J=8 & 2.5 Hz), 3.25 (s, 2H). ¹³C-NMR(MeOD, 125 MHz): δ 156.897 (C), 155.797 (C), 148.727 (CH), 145.584 (CH),138.672 (C), 137.021 (CH), 130.536 (CH), 123.476 (CH), 117.446 (C),106.221 (CH), 102.283 (CH), 32.196 (CH₂).

Hydroxylated product (20%) was also obtained and was successfullyseparated by column chromatography (See compound 11 below).

4-((2-Methoxypyridin-3-yl)methyl)benzene-1,3-diol (8)

¹H-NMR (MeOD, 500 MHz): δ 7.9015 (dd, 1H, J=1.5 & 5.0 Hz), 7.277 (dt,1H, J=1.0 & 7.0 Hz), 6.778-6.823 (m, 2H), 6.313 (dd, 1H, J=2.5 & 6.5Hz), 6.239 (dt, 1H, J=2.0 & 8.5 Hz), 3.935 (s, 3H), 3.749 (s, 2H).¹³C-NMR (MeOD, 125 MHz): δ 161.927 (C), 157.766 (C), 156.578 (C).155.941 (C), 143.115 (CH), 137.880 (CH), 130.828 (CH), 124.794 (C),116.498 (CH), 106.133 (CH), 102.098 (CH), 52.482 (CH₃), 28.505 (CH₂).

4-(2,4,6-trimethoxybenzyl)benzene-1,3-diol (9)

¹H-NMR (MeOD, 500 Hz): δ 6.587 (d, 1H, J=8.5 Hz), 6.242 (s, 1H), 6.239(s, 2H), 6.106 (dd, 1H, J=2.5 & 8.0 Hz), 3.798 (s, 3H), 3.775 (s, 6H),3.703 (s, 2H). ¹³C-NMR (CDCl₃, 125 Hz): δ 160.150 (C), 158.100 (2 C),155.986 (C), 155.509 (C), 132.723 (CH), 119.535 (C), 109.882 (C),107.232 (CH), 103.475 (CH), 91.531 (2CH), 56.258 (2 CH₃), 55.802 (CH₃),23.157 (CH₂).

4-(Benzo[d][1,3]dioxol-5-ylmethyl)benzene-1,3-diol (10)

¹H-NMR (MeOD, 500 MHz): δ 6.775 (d, 1H, J=8.5 Hz), 6.62-6.68 (m, 3H),6.295 (d, 1H, J=2.0 Hz), 6.202-6.223 (m, 1H), 5.842 (s, 2H), 3.733 (s,2H). ¹³C-NMR (MeOD, 125 MHz): δ 155.840 (C), 155.124 (C), 148.362 (C),146.548 (C), 134.234 (C), 131.868 (CH), 121.597 (CH), 119.754 (C),109.431 (CH), 108.687 (CH), 108.154 (CH), 103.761 (CH), 101.299 (CH₂),35.949 (CH₂).

4-(hydroxyl(pyridine-3-yl)methyl)benzene-1,3-diol (11)

¹H-NMR (MeOD, 300 MHz): δ 8.528 (d, 1H, J=2.1 Hz), 8.355 (dd, 1H, J=3.6& 4.8 Hz), 7.820 (td, J=1.5 & 7.5 Hz), 7.353 (t, 1H, J=5.4 Hz), 7.086(d, 1H, J=7.8 Hz), 6.315-6.267 (m, 2H), 6.067 (s, 1H); Minor partial:8.497 (d, 1H, J=2.1 Hz), 7.018 (d, 1H, J=9.3 Hz), 5.661 (s, 1H). ¹³C-NMR(MeOD, 125 MHz): δ 157.659 (C), 155.283 (C), 147.120 (CH), 146.694 (CH),141.308 (C), 135.144 (CH), 127.604 (CH), 123.495 (CH), 120.885 (C),106.466 (CH), 102.231 (CH), 68.285 (CH).

TABLE 2 Compounds Representative of Formula II Mushroom CompoundStructure Tyrosinase Assay 4-(Furan-2-ylmethyl)benzene-1,3- diol (5)

IC₅₀: = 1.8 μM 4-(Furan-3-ylmethyl)benzene-1,3- diol (6)

IC₅₀: = 3.0 μM 4-(Pyridin-ylmethyl)benzene-1,3- diol (7)

IC₅₀: = 2.0 μM 4-((2-Methoxypyridin-3- yl)methyl)benzene-1,3-diol (8)

IC₅₀: = 50.0 μM 4-(2,4,6-trimethoxybenzyl)benzene- 1,3-diol (9)

IC₅₀: = 30.0 μM 4-(B enzo[d][1,3]dioxol-5- ylmethyl)benzene-1,3-diol(10)

IC₅₀: = 0.85 μM 4-(hydroxyl(pyridine-3- yl)methyl)benzene-1,3-diol (11)

IC₅₀: = 4.9 μM

Example 3. General and Specific Methods for the Synthesis of Compoundsof Formula III

Compounds of Formula III were prepared as illustrated in Scheme 3,below. Briefly, with reference to Scheme 3, the 1,2-diphenylethenecompounds of Formula III were prepared by Wittig reaction between thecorresponding Wittig salt and aldehyde followed by hydrogenation with Pdon activated carbon and ammonium formate (AMF).

Representative Procedure for Scheme 3: 4-Phenethylbenzene-1,3-diol (12)

To a solution of the phosphonium salt of benzyl bromide (5.2 g, 12 mmol)in THE (25 mL), nBu-Li in hexane (6.9 mL, 11 mmol) was added at 0° C.The reaction mixture was allowed to stir at room temperature for 1 h,during which time the mixture developed a red color indicating theformation of ylide. A solution of 2,4-bis(benzoloxy)benzaldehyde (3.2 g,10 mmol) in THE (10 mL) was added to the ylide solution at −80° C. andallowed to stir overnight without removing the low temperature bath.During that period the red color was disappeared and a whitishsuspension was formed which was diluted with hexane and filtered. Thefiltrate was concentrated and the residue was passed through a short bedof silica gel eluting with ethyl acetate-hexane (5:95) to yield thecoupled product (3.6 g, 92%) in a ratio of (E/Z:20/80) as a pale yellowoil.

The coupled product (3 g, 7.7 mmol) was then dissolved in 24 mL THF/MeOH(1:2) followed by treatment with 10% Pd on activated carbon (300 mg) andammonium formate (3.92 g, 62 mmol) at reflux for 4 h. After cooling, thereaction mixture was filtered through celite to remove the catalyst. Thecrude product was purified by column chromatography eluting with hexaneand ethyl acetate (75:25) to yield compound 12 (1.5 g, 95%) as off-whitesolid. ¹H-NMR (MeOD, 500 MHz): δ 7.19-7.09 (m, 5H), 7.77 (d, 1H, J=8Hz), 6.35 (t, 1H, J=2.5 Hz), 6.22 (s, 1H), 2.80 (d, 2H, J=6 Hz), 2.78(d, 2H, J=4 Hz). ¹³C-NMR (MeOD, 125 MHz): δ 155.911 (C), 155.604 (C),142.535 (C), 130.302 (CH), 128.177 (CH), 128.170 (CH), 127.815 (CH),127.800 (CH), 125.238 (CH), 119.561 (C), 106.029 (CH), 102.150 (CH),36.274 (CH₂), 31.803 (CH₂).

Representative compounds 13-27 of Formula III (see Table 3) weresynthesized according to the same general reaction scheme using theappropriate staring materials. Overall yields varied from 35-55%. Insome cases, the reductive high pressure hydrogenation reaction wassuccessfully performed using Parr apparatus instead of CTH methods toobtain the final compounds.

4-(4-hydroxyphenethylbenzene)-1,3-diol (13)

¹H-NMR (MeOD, 500 MHz): δ 6.985 (dd, 2H, J=3 & 5 Hz), 6.764 (d, 1H, J=8Hz), 6.716-6.617 (m, 2H), 6.326 (d, 1H, J=2.5 Hz), 6.215 (dd, 1H, J=2.5& 8.5 Hz), 2.719 (s, 2H), 2.675 (d, 2H, J=8.5 Hz). ¹³C-NMR (MeOD, 125MHz): δ 155.756 (C), 155.500 (C), 154.560 (C), 133.707 (C), 130.354(CH), 129.125 (2CH), 119.742 (C), 114.586 (2CH), 106.048CH), 102.127(CH), 35.393 (CH₂), 31.988 (CH₂).

4-(4-methoxyphenethyl)benzene-1,3-diol (14)

¹H-NMR (MeOD, 500 MHz): δ 7.052 (d, 2H, J=8.5 Hz), 6.748-6.772 (m, 3H),6.343 (d, 1H, J=2.5 Hz), 6.222 (dd, 1H, J=2.5 & 8.0 Hz), 3.683 (s, 3H),2.745 (t, 4H, J=6.0 Hz). ¹³C-NMR (MeOD, 125 MHz): δ 157.700 (C), 155.893(C), 155.601 (C), 134.660 (C), 130.317 (CH), 129.073 (2CH), 119.608 (C),113.255 (2CH), 106.007 (CH), 102.139 (CH), 54.303 (CH₃), 35.350 (CH₂),31.944 (CH₂).

4-(2-(benzo[d][1,3]dioxol-5-ylethyl)benzene-1,3-diol (15)

¹H-NMR (MeOD, 500 MHz): δ 6.75 (d, 1H, J=8 Hz), 6.67 (d, 1H, J=8 Hz),6.66 (d, 1H, J=1.5 Hz), 6.61 (dd, 1H, J=8 & 1.5 Hz), 6.27 (d, 1H, J=2.5Hz), 6.17 (dd, 1H, J=8 & 2.5 Hz), 5.87 (s, 2H), 2.71-2.72 (m, 4H).¹³C-NMR (MeOD, 125 MHz): δ 156.01 (C), 155.01 (C), 147.41 (C), 145.47(C), 136.52 (C), 130.14 (CH), 120.87 (CH), 119.23 (C), 108.48 (CH),107.39 (CH), 105.82 (CH), 102.03 (CH), 100.45 (CH₂), 35.95 (CH₂), 32.03(CH₂).

4,4′-(ethane-1,2diyl)dibenzene-1,3-diol (16)

¹H-NMR (MeOD, 500 MHz): δ 6.81 (d, 2H, J=2 Hz), 6.29 (s, 2H), 6.20 (dd,2H, J=2 & 2 Hz), 2.68 (s, 4H). ¹³C-NMR (MeOD, 125 MHz): δ 155.83 (2 C),155.47 (2 C), 130.04 (2 CH), 145.47 (C), 120.01 (2 C), 105.95 (2 CH),102.05 (2 CH), 30.04 (2 CH₂).

4-(2,4-dimethoxyphenethyl)benzene-1,3-diol (17)

¹H-NMR (MeOD, 500 MHz): δ 6.93 (d, 1H, J=8 Hz), 6.73 (d, 1H, J=8 Hz),6.46 (d, 1H, J=2 Hz), 6.37 (dd, 1H, J=8 & 2 Hz), 6.28 (d, 1H, J=2 Hz),6.17 (dd, 1H, J=8 & 2 Hz), 3.77 (s, 3H), 3.74 (s, 3H), 2.67-2.73 (m,4H). ¹³C-NMR (MeOD, 125 MHz): δ 159.177 (C), 158.307 (C), 155.801 (C),155.564 (C), 155.564 (C), 130.047 (CH), 129.758 (CH), 122.988 (C),119.900 (C), 105.796 (CH), 103.771 (CH), 101.991 (CH), 97.867 (CH),54.392 (CH₃), 54.289 (CH₃), 29.901 (CH₂), 29.834 (CH₂).

4-(3,5-dimethoxyphenethyl)benzene-1,3-diol (18)

¹H-NMR (MeOD, 500 MHz): δ 6.77 (d, 1H, J=10 Hz), 6.34 (d, 2H, J=5 Hz),6.32 (d, 1H, J=5 Hz), 6.25 (t, 1H, J=5 Hz), 6.20 (dd, 1H, J=10 & 5 Hz),3.68 (s, 6H), 2.75 (s, 4H). ¹³C-NMR (MeOD, 125 MHz): δ 160.67 (C, 2C),155.98 (C), 155.60 (C), 144.91 (C), 130.32 (CH), 119.38 (C), 106.20 (CH,2C), 105.97 (CH), 102.08 (CH), 97.50 (CH), 54.25 (2 CH₃), 36.48 (CH₂),31.47 (CH₂).

4-(2,4-dimethoxy-3-methylphenethyl)benzene-1,3-diol (19)

¹H-NMR (MeOD, 500 MHz): δ 6.972 (d, 1H, J=8.5 Hz), 6.624 (d, 1H, J=9.0Hz), 6.285 (dd, 1H, J=1 & 2.5 Hz), 6.184 (ddd, 1H, J=1.5, 2.5 & 8 Hz),3.783 (s, 3H), 3.694 (s, 3H), 2.723-2.762 (m, 4H), 2.114 (s, 3H).¹³C-NMR (MeOD, 125 MHz): δ 157.034 (C), 156.915 (C), 155.953 (C),155.664 (C), 130.066 (CH), 127.197 (C), 126.967 (CH), 119.834 (C),118.705 (C), 105.855 (CH), 105.841 (CH), 102.046 (CH), 59.749 (CH₃),54.637 (CH₃), 31.008 (CH₂), 29.916 (CH₂), 7.941 (CH₃).

4-(2,4,6-trimethoxyphenethyl)benzene-1,3-diol (20)

¹H-NMR (MeOD, 500 MHz): δ 6.698. (dd, 1H, J=1.5 & 8 Hz), 6.262 (t, 1H,J=2.5 Hz), 6.135-6.157 (m, 3H), 3.775 (s, 3H), 3.730 (s, 3H), 3.723 (s,3H), 2.750 (t, 2H, J=8.0 Hz), 2.563 (t, 2H, J=8.0 Hz). ¹³C-NMR (MeOD,125 MHz): δ 159.369 (C), 158.799 (2C), 155.638 (C), 155.556 (C), 129.840(CH), 120.359 (C), 111.082 (C), 105.696 (CH), 101.906 (CH), 90.367 (2CH), 54.744 (2 CH₃), 54.281 (CH₃), 28.790 (CH₂), 22.653 (CH₂).

4-(1-phenylpropan-2-yl)benzene-1,3-diol (21)

¹H-NMR (MeOD, 500 Hz): δ 6.928 (d, 2H, J=8.5 Hz), 6.8541 (d, 1H, J=8.0Hz), 6.631 (d, 2H, J=8.0 Hz), 6.259 (d, 1H, J=2.0 Hz), 6.219 (dd, 1H,J=2.5 & 8.0 Hz), 3.231 (q, 1H, J=6.5 Hz), 2.854 (dd, 1H, J=5.5 & 13.0Hz), 2.519 (dd, 1H, J=9.0 & 13.5 Hz), 1.087 (d, 3H, J=7.0 Hz). ¹³C-NMR(MeOD, 500 Hz): δ 155.482 (C), 155.093 (C), 153.428 (C), 154.716 (C),132.452 (CH), 129.673 (2CH), 124.583 (C), 114.242 (2CH), 105.870 (CH),102.016 (CH), 42.284 (CH₂), 33.835 (CH), 18.575 (CH₃).

4-(2-(furan-3-yl)ethyl)benzene-1,3-diol (22)

¹H-NMR (CDCl₃, 300 MHz): δ 7.348 (s, 2H), 7.187 (s, 1H), 6.342 (d, 1H,J=8.1 Hz), 6.282 (d, 2H, J=10.2 Hz), 2.795-2.663 (m, 4H). ¹³C-NMR(CDCl₃, 75 MHz): δ 154.903 (C), 154.594 (C), 143.043 (CH), 139.307 (CH),131.236 (CH), 124.802 (C), 120.509 (C), 111.259 (CH), 107.970 (CH),103.249 (CH), 30.310 (CH₂), 25.544 (CH₂).

4-(2-(tetrahydrofuran-3-yl)ethyl)benzene-1,3-diol (23)

¹H-NMR (CDCl₃, 300 MHz): δ 6.933 (d, 1H, J=8.4 Hz), 6.359-6.283 (m, 2H),3.951-3.709 (m, 3H), 3.388 (t, 1H, J=7.5 Hz), 2.602-2.492 (m, 2H),2.262-2.163 (m, 2H), 1.707-1.585 (m, 3H). ¹³C-NMR (CDCl₃, 75 MHz): δ155.056, 154.544, 132.595, 120.639, 110.008, 107.810, 103.074, 68.188,64.108, 33.905, 32.718, 28.432.

4-(2-(furan-2-yl)ethyl)benzene-1,3-diol (24)

¹H-NMR (MeOD, 500 MHz): δ 7.31 (d, 1H, J=2 Hz), 6.78 (d, 1H, J=8 Hz),6.28 (d, 1H, J=2 Hz), 6.24 (dd, 1H, J=3 & 2 Hz), 6.18 (dd, 1H, J=8 & 2.5Hz), 5.95 (dd, 1H, J=3 Hz), 2.78-2.82 (m, 4H). ¹³C-NMR (MeOD, 125 MHz):δ 156.145 (C), 156.104 (C), 155.675 (C), 140.334 (CH), 129.995 (CH),118.790 (C), 109.576 (CH), 105.867 (CH), 104.315 (CH), 102.020 (CH),28.209 (CH₂), 28.150 (CH₂).

4-(2-(tetrahydrofuran-2-yl)ethyl)benzene-1,3-diol (25)

¹H-NMR (MeOD, 500 MHz): δ 6.83 (d, 1H, J=8 Hz), 6.26 (d, 1H, J=2.5 Hz),6.21 (dd, 1H, J=8 & 2.5 Hz), 3.79-3.85 (m, 2H), 3.70-3.73 (m, 1H),2.57-2.60 (m, 1H), 2.51-2.56 (m, 1H), 1.98-2.01 (m, 1H), 1.86-1.90 (m,2H), 1.77-1.80 (m, 1H), 1.70-1.71 (m, 1H), 1.48-1.54 (m, 1H). ¹³C-NMR(MeOD, 125 MHz): δ 156.01 (C), 155.619 (C), 155.675 (C), 129.958 (CH),119.326 (C), 105.855 (CH), 102.130 (CH), 79.187 (CH), 67.104 (CH₂),35.728 (CH₂), 30.771 (CH₂), 25.951 (CH₂), 25.218 (CH₂).

4-(2-(5-ethylofuran-2-yl)ethyl)benzene-1,3-diol (26)

¹H-NMR (MeOD, 500 MHz): δ 6.786 (d, 1H, J=8.0 Hz), 6.278 (d, 1H, J=2.5Hz), 6.1845 (dd, 1H, J=2.5 & 8.0 Hz), 5.189 (s, 2H), 2.766 (s, 4H),2.578 (q, 2H, J=7.5 Hz), 1.194 (td, 3H, J=7.5 & 1.0 Hz). ¹³C-NMR (MeOD,125 MHz): δ 156.090 (C), 155.649 (C), 155.401 (C), 154.142 (C), 129.999(CH), 118.938 (C), 105.852 (CH), 104.734 (CH), 103.779 (CH), 102.006(CH), 28.309 (CH₂), 28.268 (CH₂), 20.828 (CH₂), 11.443 (CH₃).

4-(2-(pyridin-3yl)ethyl)benzene-1,3-diol (27)

¹H-NMR (MeOD, 300 MHz): δ 8.293 (dd, 1H, J=4.8 & 1.5 Hz), 8.250 (d, 1H,J=1.8 Hz), 7.605 (dt, 1H, J=8.1 & 1.5 Hz), 7.291 (ddd, 1H, J=7.5, 4.5 &0.6 Hz), 6.673 (d, 1H, J=8.4 Hz), 6.270 (d, 1H, J=2.4 Hz), 6.139 (dd,1H, J=8.4 & 2.1 Hz), 2.880 (td, 2H, J=6.6 & 1.8 Hz), 2.778 (td, 2H,J=6.6 & 1.2 Hz). ¹³C-NMR (MeOD, 125 MHz): δ 156.06 (C), 156.04 (C),149.00 (CH), 146.00 (CH), 138.90 (C), 137.34 (CH), 130.57 (CH), 123.72(CH), 118.31 (C), 106.03 (CH), 102.25 (CH), 33.14 (CH₂), 31.48 (CH₂).

TABLE 3 Compounds Representative of Formula III Mushroom CompoundStructure Tyrosinase Assay 4-Phenethylbenzene-1,3-diol (12)

IC_(50:) = 2.8 μM 4-(4-hydroxyphenethylbenzene)-1,3- diol (13)

IC₅₀: = 2.9 μM 4-(4-methoxyphenethyl)benzene- 1,3-diol (14)

IC₅₀: = 1.2 μM 4-(2-(benzo[d][1,3]dioxol-5- ylethyl)benzene-1,3-diol(15)

IC₅₀: = 0.25 μM 4,4′-(ethane-1,2diyl)dibenzene-1,3- diol (16)

IC₅₀: = 0.35 μM 4-(2,4-dimethoxyphenethyl)benzene- 1,3-diol (17)

IC₅₀: = 3.0 μM 4-(3,5-dimethoxyphenethyl)benzene- 1,3-diol (18)

IC₅₀: = 2.8 μM 4-(2,4-dimethoxy-3- methylphenethyl)benzene-1,3-diol (19)

IC₅₀: = 0.80 μM 4-(2,4,6- trimethoxyphenethyl)benzene-1,3- diol (20)

IC₅₀: = 40.0 μM 4-(1-phenylpropan-2-yl)benzene- 1,3-diol (21)

IC₅₀: = 8.4 μM 4-(2-(furan-3-yl)ethyl)benzene-1,3- diol (22)

IC₅₀: = 1.0 μM 4-(2-(tetrahydrofuran-3- yl)ethyl)benzene-1,3-diol (23)

IC₅₀: = 5.8 μM 4-(2-(furan-2-yl)ethyl)benzene-1,3- diol (24)

IC₅₀: = 0.2 μM 4-(2-(tetrahydrofuran-2- yl)ethyl)benzene-1,3-diol (25)

IC₅₀: = 4.5 μM 4-(2-(5-ethylofuran-2- yl)ethyl)benzene-1,3-diol (26)

IC₅₀: = 0.86 μM 4-(2-(pyridin-3yl)ethyl)benzene-1,3- diol (27)

IC₅₀: = 1.8 μM

Example 4. General and Specific Methods for the Synthesis of Compoundsof Formula IV

Compounds of Formula IV were prepared as described in Scheme 4. Briefly,the 1,3-diphenylpropanes illustrative of Formula IV were prepared bybase catalyzed Aldol condensation to provide the respective chalcones,which were then reduced by catalytic transfer of hydrogen (CTH) usingammonium formate (AMF) and a Pd—C catalyst.

Representative Procedure for Scheme 4: Synthesis of Compounds 28, 29 and30 General Method for Aldol Condensation Reaction

To a solution containing 2,4-dibenzyloxyacetophenone (7.67 g, 23.1 mmol)and 3,5-dimethoxybenzaldehyde (3.80 g, 23 mmol) in 100 mL anhydrous MeOHwas added KOH (1.4 g, 25 mmol) and the mixture was stirred for 5 h atroom temperature. A yellow solid was precipitated out which was filteredand washed with water. The crude product was finally crystallized fromMeOH to afford a yellow crystalline solid (9.97 g; 87%).

General Method for CTH Reduction

The condensation product, α,β-unsaturated ketone (3 g, 7.5 mmol) wasdissolved in 24 mL glacial acetic acid followed by the addition of 10%Pd on activated carbon (300 mg) and ammonium formate (3.92 g, 62 mmol)and the reaction mixture was refluxed for 4 h. After cooling down thereaction mixture was filtered through Celite to remove the catalyst. Thecrude product was concentrated at rotary evaporator and purified bycolumn chromatography eluting with hexane and ethyl acetate (75:25) toyielded compound 28 (1.1 g, 50%) as off-white solid & compound 29 (0.57g, 25%) as thick yellow liquid.

General Method for Demethylation

To a stirred solution of 28 (1.4 g, 4.9 mmol) in dry CH₂Cl₂ (20 mL)under argon at −80° C., BBr₃ (2.67 g 10.7 mmol) was added and themixture was slowly warmed to 0° C. The reaction was quenched by addingice-cooled water and methanol was added. The solvent was then removed ina rotary evaporator and the crude product was purified by columnchromatography eluting with ethyl acetate/hexanes (25:75) to providecompound 30 (1.14 g, 90%) as a solid.

4-(3-(3,5-dimethoxyphenyl)propyl)benzene-1,3-diol (28)

¹H-NMR (MeOD, 500 MHz): δ 6.828 (d, 1H, J=8.5 Hz), 6.344 (d, 2H, J=2.5Hz), 6.288 (d, 1H, J=2.5 Hz), 2.265 (t, 1H, J=2.0 Hz), 6.226 (dd, 1H,J=2.5 Hz & 8.0 Hz), 3.723 (s, 6H), 2.503-2.558 (m, 4H), 1.800-1.826 (m,2H). ¹³C-NMR (MeOD, 125 MHz): δ 160.772 (2C), 155.860 (C), 155.597 (C),145.065 (C), 130.080 (CH), 119.774 (C), 106.055 (2 CH), 105.937 (CH),102.094 (CH), 97.326 (CH), 54.226 (2 CH₃), 35.587 (CH₂), 31.563 (CH₂),28.924 (CH₂).

4-(3-(3,5-dimethoxyphenyl)-1-hydroxypropyl)benzene-1,3-diol (29)

¹H-NMR (MeOD, 500 MHz): δ 6.977 (d, 1H, J=8.0 Hz), 6.316-6.337 (m, 3H),6.309 (t, 1H, J=2.5 Hz), 6.259 (t, 1H, J=2.5 Hz), 4.781 (dd, 1H, J=5.5Hz, J2=7.5 Hz), 3.704 (s, 6H), 2.623 (ddd, 1H, J=13.5 Hz, J2=10.0 Hz,J3=5.5 Hz), 2.5222 (ddd, 1H, J=20.0 Hz, J2=9.5 Hz, J3=6.5 Hz),2.031-1.908 (m, 2H). ¹³C-NMR (MeOD, 125 MHz): δ 160.806 (2C), 157.356(C), 156.189 (C), 144.503 (C), 128.070 (CH), 118.275 (C), 106.640 (CH),106.155 (2 CH), 102.239 (CH), 97.515 (CH), 78.440 (CH), 54.185 (CH₃),54.059 (CH₃), 37.786 (CH₂), 32.070 (CH₂).

4-(3-(3,5-dihydroxyphenyl)propyl)benzene-1,3-diol (30)

¹H-NMR (MeOD, 500 MHz): δ 6.827 (d, 1H, J=8.0 Hz), 6.267 (d, 1H, J=2.0Hz), 6.211 (dd, 1H, J=2.5 & 8.5 Hz), 6.142 (d, 2H, J=2.0 Hz), 6.074 (t,1H, J=2.5 Hz), 2.507 (t, 2H, J=8.0 Hz), 2.455 (t, 2H, J=8.0 Hz),1.76-1.84 (m, 2H). ¹³C-NMR (MeOD, 125 MHz): δ 157.848 (2C), 155.849 (C),155.582 (C), 145.065 (C), 129.995 (CH), 119.793 (C), 106.559 (2CH),105.859 (CH), 102.054 (CH), 99.514 (CH), 35.420 (CH₂), 31.593 (CH₂),28.961 (CH₂).

4-(3-(2,4,6-trimethoxyphenyl)propyl)benzene-1,3-diol (31)

Compound 31 was synthesized following a similar procedure as describedfor compound 28 above using as starting materials2,4-dibenzyloxyacetophenone and 2,4,6-trimethoxybenzaldehyde. ¹H-NMR(MeOD, 500 MHz): δ 6.822 (d, 1H, J=8.0 Hz), 6.250 (d, 1H, J=2.5 Hz),6.197 (dd, 1H, J=2.5 & 8.0 Hz), 6.165 (s, 2H), 3.768 (s, 3H), 3.764 (s,6H), 2.562 (t, 2H, J=7.5 Hz), 2.461 (t, 2H, J=7.5 Hz), 1.617-1.653 (m,2H). ¹³C-NMR (MeOD, 125 MHz): δ 159.273 (C), 158.729 (2C), 155.556 (C),155.449 (C), 129.655 (2CH), 120.426 (C), 111.382 (C), 105.789 (CH),101.968 (CH), 90.285 (CH), 54.655 (2CH₃), 54.274 (CH₃), 29.716 (CH₂),29.064 (CH₂), 22.142 (CH₂).

4-(3-(2,4-dihydroxyphenyl)propyl)-2-methylbenzene-1,3-diol (32)

Compound 32 was synthesized following a similar procedure as describedfor compound 30 using as starting materials 2,4-dibenzyloxyacetophenoneand 2,4-dimethoxy-3-methylbenzaldehyde. ¹H-NMR (MeOD, 500 MHz): δ 6.879(d, 1H, J=8.5 Hz), 6.747 (d, 1H, J=8.0 Hz), 6.352 (d, 1H, J=1.5 Hz),6.341 (d, 1H, J=4.0 Hz), 6.285 (dd, 1H, J=8.0 Hz & 2.5 Hz), 2.567 (dd,4H, J=8.0 & 7.5 Hz), 2.133 (s, 3H), 1.786-1.817 (m, 2H). ¹³C-NMR (MeOD,125 MHz): δ 155.486 (C), 155.316 (C), 153.428 (C), 152.906 (C), 130.295(CH), 126.523 (CH), 120.933 (C), 120.518 (C), 111.556 (C), 106.788 (CH),106.377 (CH), 102.283 (CH), 30.767 (CH₂), 29.649 (CH₂), 29.127 (CH₂),7.833 (CH₃).

4-(3-(2,4-dihydroxy-3-methylphenyl)-3-(2,4-dihydroxyphenyl)propane-1-one(33)

Compound 33 was synthesized following a similar procedure as describedfor compound 28 using as starting materials 2,4-dibenzyloxyacetophenoneand 2,4-dimethoxy-3-methylbenzaldehyde. The reduction with Pd—C/ammoniumformate was carried out at room temperature instead of refluxing.Demethylation reaction with borontribromide was performed using assimilar protocol described as in the procedure for compound 30. ¹H-NMR(MeOD, 500 MHz): δ 7.628 (d, 1H, J=9.0 Hz), 6.868 (d, 1H, J=8.0 Hz),6.349 (d, 1H, J=9.0 Hz), 6.283 (d, 1H, J=2.5 Hz), 6.203 (dd, 1H, J=2.5 &8.0 Hz), 3.127 (t, 2H, J=7.5 Hz), 2.862 (t, 2H, J=7.5 Hz), 2.031 (s,3H). ¹³C-NMR (MeOD, 500 Hz): δ 205.420 (C), 162.894 (C), 162.264 (C),156.438 (C), 155.782 (C), 130.710 (CH), 129.510 (CH), 118.364 (C),112.249 (C), 110.820 (C), 106.596 (CH), 105.970 (CH), 102.142 (CH),47.089 (CH₂), 25.810 (CH₂), 6.361 (CH₃).

Example 5. Synthesis of 4-(5-Hydroxyphenyl)Benzene-1,3-Diol (34)

Compound 34 was synthesized as set forth in Scheme 5

To a solution of the phosphonium salt of 2.4-bis(benzoloxy)benzylbromide (7.72, 12 mmol) in THE (25 mL) was added nBu-Li in hexane (6.9mL, 11 mmol) at 0° C. The reaction mixture was allowed to stir at roomtemperature for 1 h, during that period the mixture developed a redcolor indicating the formation of ylide. A solution of4-(tetrahydropyranyloxy)butanal (1.72 g, 10 mmol) in THE (10 mL) wasadded to the ylide solution at −80° C. and the mixture was allowed tostir overnight without removing the low temperature bath, during thatperiod the red color disappeared and a whitish suspension was formedwhich was diluted with hexane and filtered. The filtrate wasconcentrated and residue was passed through a short bed of silica geleluting with ethyl acetate-hexane (5:95) to give the coupled product(5.2 g, 92%) in a ratio of (E/Z:20/80) as pale yellow oil.

The coupled product (3.6 g, 7.7 mmol) was then dissolved in 24 mLTHF/MeOH (1:2) followed by treatment with 10% Pd on activated carbon(360 mg) and ammonium formate (3.92 g, 62 mmol) at reflux for 4 h. Aftercooling down, the reaction mixture was filtered through celite to removethe catalyst. The crude product, after concentration, was purified bycolumn chromatography eluting with hexane and ethyl acetate (80:20) toyield the reduced product (2.15 g, 95%) as a colorless thick liquid.

To a stirred solution of the reduced product (1.2 g, 4 mmol) in ethanol(20 mL) was added pyridinium toluene-4-sulfonate (350 mg, 1.39 mmol) atroom temperature and the mixture was stirred for 24 h. Removal of thesolvent in vacuum followed by column chromatography of the residue onsilica gel eluting with ethyl acetate-hexane (10:90) gave the titlecompound (34) as off-white solid (690 mg, 88%). ¹H-NMR (MeOD, 500 MHz):δ 8.825 (d, 1H, J=7.5 Hz), 6.2705 (d, 1H, J=0.5 Hz), 6.215 (d, 1H, J=7.5Hz), 3.538 (t, 2H, J=6.5 Hz), 2.491 (t, 2H, J=7.5 Hz), 1.526-1.585 (m,4H), 1.358-1.40 (m, 2H). ¹³C-NMR (MeOD, 125 MHz): δ 155.705 (C), 155.482(C), 129.984 (CH), 120.108 (C), 105.926 (CH), 102.061 (CH), δ 1.722(CH₂), 32.189 (CH₂), 29.182 (CH₂), 29.086 (CH₂), 25.322 (CH₂), 103.761(CH).

TABLE 4 Compounds Representative of Formula IV Mushroom CompoundStructure Tyrosinase Assay 4-(3-(3,5-dimethoxyphenyl)propyl)benzene-1,3- diol (28)

IC₅₀: = 1.02 μM 4-(3-(3,5-dimethoxypheny1)-1-hydroxypropyl)benzene-1,3-diol (29)

IC₅₀: = 5.15 μM 4-(3-(3,5- dihydroxyphenyl)propyl)benzene-1,3- diol (30)

IC₅₀: = 0.5 μM 4-(3-(2,4,6- trimethoxyphenyl)propyl)benzene-1,3- diol(31)

IC₅₀: = 0.165 μM 4-(3-(2,4-dihydroxyphenyl)propyl)-2-methylbenzene-1,3-diol (32)

IC₅₀: = 2.3 μM 4-(3-(2,4-dihydroxy-3-methylphenyl)-3-(2,4-dihydroxyphenyl)propane-1-one (33)

IC₅₀ = 2.54 μM 4-(5-hydroxypentyl)benzene-1,3-diol (34)

IC₅₀ = 1.0 μM

Example 6. Tyrosinase Assay Reagents

Tyrosinase isolated from the mushroom species Agaricus bisporus waspurchased from the Sigma-Aldrich Inc. (Cat #T3824-50KU). The enzyme wasdissolved in the tyrosinase assay buffer (100 mM sodium phosphate, pH6.8) to a concentration 10 U/μl, and stored at −70° C. For theexperiments the enzyme was freshly diluted in the assay buffer to aconcentration of 0.2 U/μl.

All test compounds were initially dissolved in 100% DMSO at aconcentration of 400 mM. The compounds were further diluted in 100% DMSOto a concentration of 80 μM. 80 μM stock solutions of the compounds werethen diluted ten times in the tyrosinase assay buffer to theconcentration of 8 μM. The compounds were then serially diluted atthree-fold increments in the assay buffer containing 10% DMSO, keepingthe concentration of DMSO constant across all samples. These seriallydiluted compounds were subsequently used as two-fold concentrated stocksolutions in the tyrosinase activity assays.

Tyrosinase substrate, L-DOPA (Sigma-Aldrich Inc, Cat #37830) wasdissolved in the tyrosinase assay buffer to a concentration of 4 mM.This solution was then used as the four-fold concentrated stock ofsubstrate in the tyrosinase activity assays.

Assay Conditions

Tyrosinase assays were performed in clear-bottom 96-well plates at roomtemperature. The final volume of the assays was 200 μl per well. 100 μlof two-fold concentrated test compounds were mixed with 50 μl of 4 mML-DOPA. The reactions were initiated by adding 50 μl of mushroomtyrosinase (0.2 U/μl, 10 U per reaction), and allowed to proceed for 15minutes. Accumulation of colored product was monitored by lightabsorption at 450 nm using Victor 2 plate reader (Perkin-Elmer Inc.).

Results

The assays were performed in triplicate and covered a concentrationrange of test compounds from 4 μM to 5.5 nM. Mean (n=3) absorption ofwells containing no enzyme was subtracted as blanks. The data wascomputed as percentage activity of wells that contained the tyrosinase,but no test compounds. IC₅₀ values were computed from non-linearregression fits using GraphPad Prism software. The results forrepresentative compounds of Formulas I-IV are set forth in Tables 1-4below, and FIGS. 1-9 .

Example 7. Murine Melanoma Cell-Based Assays

Selected compounds were then tested for the ability to suppress melaninproduction by the murine melanoma cells B16-F1 as detailed below.

Materials

Murine melanoma cells B16-F1 were purchased from the ATCC (Cat#CRL-6323). CellTiter96 AqueousOne Solution was purchased from Promega(Cat #G3581). 0.2 μm pore size, low protein binding filters werepurchased from PALL Life Sciences. (Cat #PN4454). Alpha-MSH waspurchased from the Bachem Inc. (Cat #H-1075.0005). All tissue culturereagents were purchased from the Invitrogen Inc.

The B16-F1 cells were maintained in cell growth media (DMEM/High Glucosesupplemented with glutamine, sodium pyruvate, 10% dialyzed fetal calfserum, 1% non-essential amino acids, 50 units/mL penicillin, and 50μg/mL of streptomycin) at 37° C. in a humidified atmosphere of 95% airand 5% CO₂.

All test compounds were initially dissolved in 100% DMSO at aconcentration of 400 mM. Then, 6 μl of 400 mM test compounds were addedto 1.2 mL of the cell growth media supplemented with 200 nM alpha-MSH,giving a final concentration of 2 mM for the test compound andconcentration of 0.5% for DMSO. The compounds were centrifuged for 1 hat 20,000×g. Supernatants (1 mL) were collected and filtered throughsterile 0.2 μm filters. The compounds were serially diluted in two-foldincrements in the sterile cell growth media supplemented with 0.5% DMSOand 200 nM alpha-MSH, thus keeping the concentration of alpha-MSH andDMSO constant for all samples. These serially diluted compounds weresubsequently used as two-fold concentrated stock solutions in themelanin production and cell viability assays.

Methods

B16-F1 cells were seeded into the wells of clear-bottom 96-well platesat 40,000 cells per well, in 100 μl of the cell growth media. On thefollowing day, 100 μl of freshly prepared two fold concentrated testcompounds were added to the wells. The cells were maintained at 37° C.in a humidified atmosphere of 95% air and 5% CO₂ for 72 hours. At theend of the incubation period melanin-containing conditioned media wasremoved from the cells and transferred to another plate. To measuremelanin content the light absorbance of conditioned media was taken at450 nm using a Victor 2 plate reader (Perkin-Elmer Inc.).

Viability of cells was measured using standard tetrazolium reductionassay based on redox potential of live cells. After aspiration ofmelanin-containing conditioned media, the cells were immediatelyreplenished with 100 μl of fresh media supplemented with 16% CellTiter96AqueousOne solution containing tetrazolium salts. The cells weremaintained at 37° C. for additional 20-40 minutes. Conversion oftetrazolium was monitored by measuring absorbance of cell wells at 450nm using a Victor 2 plate reader (Perkin-Elmer Inc.

Results

The results of the representative experiments are summarized in Table 5and FIGS. 1-9 . More than 90% percent of the melanin produced bycultured melanoma cells is found in extra-cellular media. Therefore, atthe end of the experiment melanin-containing media was collected andrelative amounts of melanin were determined by adsorption at 450 nm.Viability of cells was determined by a commonly used colorimetricprocedure that is based on conversion of the tetrazolium compounds tocolored formazan products (using Promega's CellTiter96 AqueousOneassay). Dehydrogenase enzymes in metabolically active cells accomplishthis conversion, and the amount of formazan product is directlyproportional to the number of living cells in culture.

The assays were performed in quadruplicate and covered a concentrationrange of test compounds from 2 μM to 1000 μM. Mean (n=4) absorption ofwells containing no cells was subtracted as blanks. The results werecomputed as percent of wells that contained the cells but no testcompounds. IC₅₀ values were computed from non-linear regression fitsusing GraphPad Prism software. The results are set forth in Table 5.

TABLE 5 Results of Mushroom Tyrosinase Assay, Melanin Production Assayand Cell Viability Studies for representative compounds of Formulas I-IVMushroom Melanin Compound Structure Tyrosinase Assay production Cellviability  7

IC₅₀: = 2.0 μM IC₅₀: = 14 μM 15

IC₅₀: = 0.25 μM IC₅₀: = 3.3 μM LD₅₀: = 66 μM 16

IC₅₀: = 0.35 μM IC₅₀: = 1.7 μM LD₅₀: = 260 μM 17

IC₅₀: = 3.0 μM IC₅₀: = 11.9 μM LD₅₀: = 157.8 μM 18

IC₅₀: = 2.8 μM IC₅₀: = 2.97 μM LD₅₀: = 222.2 μM 19

IC₅₀: = 0.80 μM IC₅₀: = 24.7 μM LD₅₀: = 130.3 μM 24

IC₅₀: = 0.2 μM IC₅₀: = 1.60 μM LD₅₀: = 345.9 μM 25

IC₅₀: = 4.5 μM IC₅₀: = 5 μM LD₅₀: >1000 μM 31

IC₅₀: = 0.165 μM IC₅₀: = 156.1 μM LD₅₀: = 187.8 μM kojic acid

IC₅₀: = 20 μM IC₅₀: = 303.5 μM LD₅₀: = >1000 μM

Example 8. Reconstructed Human Skin Studies Materials and Methods

The skin whitening effects of test compounds were studied using areconstructed skin model, Melanoderm™ provided by MatTek Corp. (Ashland,MA) according to the manufacturer's specifications. Briefly, normalhuman epidermal keratinocytes and normal human melanocytes derived fromdark skin donors were co-cultured on a surface of collagen-coatedmembrane to form multi-layered, highly differentiated skin tissue(MEL-300-B). The tissues were maintained in the CO₂ incubator at 37° C.The apical surfaces of the reconstructed skin (9 mm in diameter) wereexposed to air whereas the bottom surfaces remained in contact with 5 mLof maintenance medium, containing skin differentiating factors(EPI-100-NNM-113). Test compounds were formulated in 80% propyleneglycol as follows: 10 mg of each test compound was dissolved overnightin 1 mL of propylene glycol (1,2-propanediol, Sigma-Aldrich). Thecompounds were then sterilized by passing through 0.2 μm filter anddiluted in sterile water/propylene glycol to final concentrations 0.2%.0.4% and 0.8%. The concentration of propylene glycol was kept 80% forall samples. In addition the following controls were used: sterilewater, 80% propylene glycol, and 1% kojic acid in water.

Test compounds were applied to the apical surface of the tissues asfollows: 10 μl of each test compound, 10 μl of 80% propylene glycol(vehicle control), 25 μl of sterile water (negative control) and 25 μlof 1% kojic acid (positive control). The samples were re-applied everyother day for 15 days. All the samples were tested in duplicate. At theend of the experiment microscopic images were taken usingphoto-microscope equipped with 25× phase contrast objective and colorCCD camera.

Results

The potential skin-whitening properties of test compounds were furtherexplored in a reconstructed skin model. The model consists of normal,human-derived epidermal keratinocytes and melanocytes, which have beenco-cultured to form a multilayered, highly differentiated humanepidermis. In this study the melanocytes were obtained from a highlypigmented donor. The reconstructed skin was grown at the air-liquidinterface, making it possible to mimic topical application of skinwhitening agents (FIG. 10A).

Different concentrations of test compounds, 80% propylene glycol (thevehicle control), water (negative control) or 1% kojic acid (positivecontrol) were repeatedly applied topically on the surface of thereconstructed skin for 15 days. Two of the test compounds, namely,compound #16, and compound #25 exhibited significant whitening effectson skin melanocytes, without causing any detectable alterations of cellmorphology (FIG. 10B). Of them, compound #16 exhibited the greatesteffect with significant whitening of melanocytes observed as early as 3days after the beginning of the experiment (data not shown). Photographsof skin specimen taken after 15 days of the experiment show significantdose-dependent whitening effects on melanocytes, which appear onphotographs as dark dendritic cells.

1-65: (canceled)
 66. A compound of Formula V:

or a pharmaceutically acceptable salt thereof, wherein: R₁ is selectedfrom the group consisting of H or C₁-C₁₀ alkyl; Bn is a benzyl; and R′is 1 to 3 moieties (R′, R″, R′″) independently selected from the groupconsisting of a C₁-C₁₀ alkyl group, a C₁-C₁₀ alkoxy group or a hydroxylgroup.
 67. A composition comprising the compound of claim 61 and apharmaceutically or cosmetically acceptable carrier.
 68. A method ofproducing the compound in claim 61 by utilizing a compound comprisingFormula IV as a starter compound or an intermediate compound:

wherein

represents double or single bond, R₁ is selected from the groupconsisting of H or C₁-C₁₀ alkyl; R₄ is selected from the groupconsisting of ═H, OH or O; and R₅ is a selected from the groupconsisting of a substituted or unsubstituted: aromatic ring,heteroaromatic ring, heterocyclic ring or a hydroxylalkyl moiety. 69.The method of claim 68, wherein R₁ is H or CH₃ and R₅ is selected from asubstituted or unsubstituted: aromatic ring, heteroaromatic ring or aheterocyclic ring or a C₁-C₁₀ hydroxylalkyl (—C₁-C₁₀)—OH).
 70. Themethod of claim 68, wherein R₅ is substituted with 1 to 3 moieties (R′,R″, R′″) independently selected from the group consisting of a C₁-C₁₀alkyl group, a C₁-C₁₀ alkoxy group or a hydroxyl group.